Calling Clonotypes

Calling method from contigs

1. Using Gene information

• VDJC(TRA/TRB)
  
• CDR3nt
  
• CDR3aa
  
• Combination

2. Using Chain information

• TRA1+TRB1
• TRA1 or TRB1
• multi-chains
  

Basic Clonotype Analysis

Immune repertoire analysis

1. Diversity Index

• Shannon Entropy
• Simpson

2. Repertoire analysis.

• TRAV/D/J/C distribution
• TRBV/D/J/C distribution
• CDR3nt length distribution
• Clustering of clonotype repertoire

3. Expansion study

• Clonotype expansion (hyper,high,medium,small,rare)
• Top 100 expanded clonotypes with TRAV:TRBV info

Target Cell:Clonotype Analysis

Combination with scRNA-seq (if available)

1. Target cell selection

• Cells with target features (e.g. cytotoxic cells, effector T cells, NKT, etc).
• Cells associated with expanded clonotypes (hyper, high, medium, small, rare).
• Cells with clonotypes displaying treatment-specific response.

2. Correlation of clonotype frequency with related data

• Correlation of clonotype frequency with target gene expression profiles.
  
• Correlation with clinical outcomes and phenotypic data. (if available)

3. Comparative Analysis

• Comparison between different patient groups (e.g., responders vs non-responders to therapy, different disease stages) and associated clonotypes.
• Clonotype repertoires to identify conserved features of the immune response.

4. Statistical and Computational Approaches

• Application of statistical models and computational algorithms for the analysis.
  
• Evaluation of the robustness and reproducibility of findings.

Clonotype Distribution

Cytotoxic Gene Expression Profiling across Clonotypes and Samples

Important Cytotoxic Gene Information

NKG7
- Natural Killer Growth Factor 7, the function of cytotoxic lymphocytes, particularly Natural Killer (NK) cells, potentially through the regulation of apoptotic pathways.

KLRG1
- Killer Cell Lectin-Like Receptor G1, is a transmembrane protein receptor primarily expressed on cytotoxic lymphocytes, regulating their activation and effector functions.

IFNG
- Interferon Gamma, is a cytokine crucially involved in the activation and effector functions of cytotoxic lymphocytes, including NK cells and cytotoxic T cells. IFNG enhances the cytotoxicity of these cells, promotes antigen presentation, and directs T helper cell differentiation towards a Th1 phenotype, crucial for effective immune responses.

GZMA
- Granzyme A, is a serine protease predominantly expressed in cytotoxic lymphocytes, facilitating target cell apoptosis.Upon recognition of target cells, GZMA is released and initiates caspase-independent cell death pathways, leading to the elimination of infected or transformed cells.

PRF1
- Perforin-1, is a pore-forming protein primarily expressed in cytotoxic lymphocytes, essential for target cell lysis. PRF1 forms pores in target cell membranes, enabling the delivery of pro-apoptotic molecules and promoting target cell death, crucial for immune surveillance and defense.

Heatmap